Substantial evidence suggests that high glucose (HG) causes endothelial cell damage; however, the potential mechanism therein has yet to be clarified. The aim of this study was to investigate the influence of HG on the endothelial-to-mesenchymal transition (EndMT) and its relevance to the activation of the renin-angiotensin system.Primary human aortic endothelial cells (HAECs) were divided into three groups: a normal glucose (NG) group, HG group, and irbesartan (1 μM)-treated (HG+irbesartan) group. The concentration of angiotensin II in the supernatant was detected by radioimmunoassay. Pathological changes were investigated using fluorescence microscopy and electron microscopy. Immunofluorescence staining was performed to detect the co-expression of CD31 and fibroblast markers, such as fibroblast-specific protein 1 (FSP1). The expressions of FSP1 and α-SMA were detected by RT-PCR and Western blot.P < 0.05). Double staining of the HAECs indicated a co-localization of CD31 and FSP1 and that some cells acquired spindle-shaped morphologies and a loss of CD31 staining; however, treatment with irbesartan attenuated the expression of EndMT (P < 0.05).The treatment of HAECs in the HG group resulted in significant increases in the expressions of FSP1 and angiotensin II in dose-and time-dependent manners. The incubation of HAECs exposure to HG resulted in a fibroblast-like phenotype, wherein increased microfilamentation and a roughened endoplasmic reticulum structure were observed in the cytoplasm. The expressions of FSP1 and α-SMA were significantly increased in the HG group, and these changes were inhibited by irbesartan treatment (These findings suggest a novel mechanism in HG-induced endothelial damage via the mediation of the EndMT by angiotensin II, which was inhibited by Irbesartan. Vascular complications, such as cardiomyopathy and nephropathy, are the leading cause of morbidity and mortality in patients with diabetes. Because the initial injury by hyperglycemia occurs in the blood vessels, endothelial cells are considered to be the first target, and, furthermore, endothelial damage plays an important role in the development and progression of diabetic vascular complications -3. Four Recent studies have indicated that the endothelial-to-mesenchymal transition (EndMT) could contribute to the progression of diabetic nephropathy, diabetic renal fibrosis, and cardiac fibrosis -7, and tHAECs were purchased from Sciencell (No. 6100) and grown in a Sciencell endothelial basal medium . This ECM consists of 500 ml of basal medium, 25 ml of fetal bovine serum (No. 0025), 5 ml of endothelial cell growth supplement (No. 1052), and 5 ml of a penicillin/streptomycin solution (No. 0503).2. The medium was changed every other day until the culture was approximately 50% confluent. When the culture reached 50% confluence, the medium was changed every day until the culture was approximately 80% confluent. HAECs were performed between the 2-4 passages. The culture medium was changed to a serum-free solution for 24 h, and the HAECs were treated with normal glucose , HG (15 mM or 30 mM D-glucose) [Cells were cultured at 37°C in a humidified atmosphere with 5% COglucose) , or 5.5 glucose)  for 48 hAng II was measured in the supernatant by radioimmunoassay, as previously described . A commeTotal RNA was prepared from the HAECs using TRIzol (Key GEN). Total RNA was prepared using TRIzol (Key GEN) from HAEC. PCR reactions were performed using specific primer pairs: a FSP1 sense primer: 5' TTGGGGAAAAG GACAGATGAAG 3', anti-sense primer: 5'TGAAGGAGCCAGGGTGGAAAAA 3'), α-SMA sense prime: 5'ATAACATCAAGCCCAAATCTGC3', anti-sense primer: 5' TTCCTTTTTTCTTTCCCAACA 3') and a GADPH sense primer: 5'AAGGTCG GAGTCAACGGATTT 3', antisense primer: 5'AGATGATGACCCTTTTGGCTC 3').Equal amounts of cell lysate proteins (30 μg) were separated on 4-20% SDS-polyacrylamide gels and transferred onto nitrocellulose membranes .The membranes were incubated overnight with polyclonal rabbit anti-rat FSP1 and the polyclonal rabbit anti-rat α-SMA , followed by a horseradish peroxidase-labeled goat anti-rabbit IgG . The signals were detected using an ECL advance system .+ cells were observed to have oval and elongated shapes in the HG group. The pictures were captured by the LSM5 image browser (Zeiss) and analyzed using a laser scanning confocal microscope .For a double immunofluorescence procedure, we incubated the HAECs with two primary antibodies at 4°C overnight. The primary antibodies were monoclonal mouse anti-CD31  and polyclonal rabbit anti-FSP1 . We incubated cells in 1% BSA for 1 h at room temperature in the dark with a mixture of two secondary antibodies and two different fluorochromes: Rhod red-conjugated goat anti-rabbit and FITC green-conjugated goat anti-mouse. As a negative control, the primary antibody was replaced with non-immune IgG, and no staining could be observed. FSP1Ultra-thin cells were counter-stained with uranyl acetate and lead citrate and were examined with a transmission electron microscope . The LSM5 image browser (Zeiss) was used to capture images of morphological changes in the HAECs using CD31 immunofluorescence staining, as previously mentioned.P < 0.05.Data were expressed as mean ± standard deviation (SD) and analyzed by one-way analysis of the variance (ANOVA) using SPSS, version 13.0. Data were considered significant if To demonstrate that enhanced Ang II production depended on the concentration and duration of HG exposure, we incubated HAECs in a medium that contained 5.5, 15, or 30 mM glucose for 48 h. Mannitol was added to the control cell incubation medium to equalize the osmolarity. Ang II was observed to increase in a dose-dependent manner in response to HG exposure Fig. . The conP < 0.05).As shown in Fig. #P < 0.05 vs. NG), reaching a peak at 30 mM HG with a 15.62-fold increase in comparison to that with NG exposure .According to Fig. ure Fig. . In respP < 0.05). In the control cells, FSP1 expression was confined to sparsely scattered fibroblasts  and fibroblast markers FSP1 . Confocal microscopy revealed the co-localization of both FSP1 and CD31 Fig. . An analsts Fig. .Normal endothelial monolayers displayed a typical cobblestone morphology. We observed that HAECs exposure to HG for 48 h exhibited profound changes with cells becoming elongated, spindle-shaped and lost cobblestone morphologies according to fluorescence microscopic analysis Fig. . InteresElectron microscopy analysis of the control demonstrated that the endothelial cells therein exhibited normal structures Fig. , × 6000.Microangiopathy is the most common complication in diabetes, wherein endothelial cell injury is an early feature of microvascular lesions. Studies have shown that endothelial injury accelerates atherosclerosis and subsequently causes cardiovascular events . EmerginAlthough the EndMT was first investigated as a critical process in heart development , it is nHow did HG induce EndMT? In our study, we observed that irbesartan as an ARB significantly inhibited the EndMT. Furthermore, other studies have demonstrated the antiproteinuric effects and the preservation of endothelial function that derive from ARB, which translate into cardiovascular and renoprotective benefits that extend beyond the lowering of blood pressure ,27. In vThese findings suggest a novel and early mechanism concerning HG-induced endothelial damage via an angiotensin II-mediated EndMT, which provides new insight into the early application of ARB in the protection of blood vessels and the prevention of organ failure in diabetes.EndMT: endothelial-to-mesenchymal transition; HAECs: human aortic endothelial cells; HG: high glucose; Irb: Irbesartan; FSP1: fibroblast-specific protein1; AGEs: advanced glycation end-products; RAS: rennin-angiotensin system; Ang II: angiotensin II; ARB: angiotensin II receptor type 1 blocker.The authors declare that they have no competing interests.TR performed the experiments, analyzed data, interpreted results, and wrote the manuscript. LQ participated in the HAEC culture and analysis. LL and DH carried out the RT-PCR and Western blotting. ZM helped to carry out the immunofluorescent staining. MK coordinated the study and was involved in the data interpretation. LB participated in the study design and coordination and helped review the manuscript. All authors read and approved the final manuscript.


Glioblastoma multiforme (GBM) is resistant to current cytotoxic therapies, in part because of enhanced DNA repair. Activation of the receptor tyrosine kinase c-Met has been shown to protect cancer cells from DNA damage. We hypothesized that inhibiting c-Met would decrease this protection and thus sensitize resistant tumor cells to the effects of radiation therapy.in vivo radiosensitization studies.Eight human GBM cell lines were screened for radiosensitivity to the small-molecule c-Met inhibitor MP470 with colony-count assays. Double-strand (ds) DNA breaks was quantified by using antibodies to gamma H2AX. Western blotting demonstrate expression of RAD51, glycogen synthase kinase (GSK)-3β, and other proteins. A murine xenograft tumor flank model was used for In vivo, the addition of MP470 to radiation resulted in a tumor-growth-delay enhancement ratio of 2.9 over radiation alone and extended survival time.MP470 reduced c-Met phosphorylation and enhanced radiation-induced cell kill by 0.4 logs in SF767 cells. Cells pretreated with MP470 had more ds DNA damage than cells treated with radiation alone. Mechanistically, MP470 was shown to inhibit dsDNA break repair and increase apoptosis. MP470 influences various survival and DNA repair related proteins such as pAKT, RAD51 and GSK3β. in vitro and in vivo, and may help to improve outcomes for patients with GBM.GBM is a disease site where radiation is often used to address both macroscopic and microscopic disease. Despite attempts at dose escalation outcomes remain poor. MP470, a potent small-molecule tyrosine kinase inhibitor of c-Met, radiosensitized several GBM cell lines both  The management of malignant gliomas continues to pose a difficult therapeutic challenge. Use of radiation therapy and chemotherapy after maximal tumor debulking can improve both local control and survival for some patients with malignant gliomas . UnfortuHepatocyte growth factor (HGF) is a multifunctional heterodimeric protein typically produced by mesenchymal cells. Its pleiotropic activities are mediated through its cellular receptor, a transmembrane tyrosine kinase encoded by the proto-oncogene c-Met. In malignant cells, HGF has been shown to protect cells from death induced by a variety of DNA-damaging agents, including radiation and topoisomerase inhibitors . Interesin vitro and in vivo.In the work presented here, a novel inhibitor of c-Met tyrosine kinase, MP470 , was tes2 incubator. MP470  was stored in the dark at 4°C until use, when it was dissolved in dimethyl sulfoxide and used at a final concentration of 5.0-10 μM. The drug was added to cells 1 hour before irradiation unless otherwise specified. Control cells were treated with equal volumes of dimethylsulfoxide. A cobalt-60 teletherapy unit (Atomic Energy of Canada Limited Theratron-80) was used to irradiate the GBM cells at a dose rate of 2 Gy/min.All of the human GBM cell lines tested  were obtained from the University of California, San Francisco, and maintained in Dulbecco's Modified Eagle Medium supplemented with 10% fetal calf serum and 1% penicillin-streptomycin   [Cells were plated on 10-cm petri dishes and grown for 24-48 hours. MP470 was then added at a concentration of 10 hnology) .siRNA to c-Met (sc-29397) and control siRNA (sc-37007) were purchased from Santa Cruz Biotechnology. The transfection reagent Lipofectamine was from Invitrogen . U87 cells were grown to 70% confluence and transfected with siRNA at a final concentration of 100 nM. Seventy-two hours later, the cells were lysed for western blotting analysis as described above.To create subcutaneous tumors, cells  were implanted in the flanks of 32 outbred athymic nude mice, 8 per arm . U87 cells were chosen for their high level of c-Met expression and ability to rapidly produce tumors. Twenty-five days after the cells were injected, animals were pair-matched (based on tumor volume) and assigned to one of four treatment groups: control; MP470 (60 mg/kg) alone; radiation alone (2 Gy per day × 10 days); and MP470 + radiation. MP470 was delivered daily by gavage at a dose of 60 mg/kg in peanut oil starting on day 25 for 14 consecutive days. Radiation was started on day 27 and consisted of 2 Gy per day delivered to the tumor by a cobalt-60 irradiator (Theratron-80).2 asphyxiation when the tumor volume reached 2000 mm3, as required by our institutional animal care and use committee protocol #07-029. All remaining animals were euthanized on day 48. Tumors were measured with calipers every 5 days and the volume calculated according to the formula (a2 - b/2), where a is the smallest diameter and b is the largest diameter of the tumor.Radiation was delivered daily, 5 days per week for 2 weeks, at 1 hour after the MP470 treatment. The total cumulative dose delivered to the tumor was thus 20 Gy. Animals were euthanized by CO3 minus the number of days for tumors in the control group to reach the same size. Normalized growth delay (NGD) was calculated as the number of days for tumors in the combined-therapy group to reach 1,500 mm3 minus the number of days for tumors in the MP470-only group to reach 1,500 mm3. The enhancement factor was then determined by dividing the NGD for the group receiving MP470 plus radiation by the AGD for the group given radiation alone. All statistical analyses were carried out with Stata 9.2 for Windows , and P values < 0.05 were considered significant.Tumor growth delay was expressed in absolute and normalized terms as follows. Absolute growth delay (AGD) was defined as the number of days for tumors in the radiation-only and the MP470 + radiation groups to reach 1,500 mm50 values ranging from 1 μM to 10 μM (median 5 μM). To test its potential as a radiosensitizer, we assessed clonogenic survival after 4 Gy of the same eight GBM cell lines after a 1-hour treatment with MP470 followed by a single radiation dose , for 8-Gy irradiation alone and 35 ± 4.3 tail DNA (%), for MP470 followed by 8-Gy irradiation). This increase in OTM suggests that MP470's radiosensitizing effect may be partially mediated through inhibition of dsDNA repair. RAD51 is a key regulator of homologous recombinational repair [μM. To verify that MP470 was indeed decreasing RAD51 expression and not simply shifting cells into a quiescent cell cycle state (G0) characterized by lower levels of RAD51, we tested the effect of MP470 on cell cycle distribution and found it had no influence. To establish that RAD51 suppression was directly associated with c-Met inhibition, we silenced c-Met expression using siRNA, which also demonstrated inhibition of RAD51 , and 87.5% in the MP470-plus-radiation group (P = 0.001)  Fig. .in vitro [in vitro findings were supported by our in vivo observations using a xenograft model in nude mice. In this model, MP470 by itself, at a dose of 60 mg/kg, had no effect on tumor size or survival; radiation by itself was somewhat more effective in reducing tumor volume and improving survival; but the combination of radiation plus MP470 produced the best response in terms of both local control and survival.The small molecule MP470 is a potent c-Met antagonist that is cytotoxic to a variety of cell lines in vitro . In thisHigh-grade glial neoplasms of the brain continue to be one of the most challenging malignancies to treat, and their poor prognosis has improved only marginally over the past four decades  . To bettin vitro using a retrovirally based approach that would not be a clinically viable option, although it did serve as an important proof of concept [To our knowledge, this is the first demonstration that MP470, an orally available c-Met antagonist, causes radiosensitization of several GBM cell lines. We have shown evidence that supports a mechanism of action consistent with a decrease in dsDNA break repair, along with enhanced radiation-induced apoptosis. Other investigators have shown that c-Met inhibition can enhance radiation-induced tumor cell death  concept . This st concept . This inJames Welsh, Research support from SuperGen, holds patent on MP-470Daruka Mahadevan, Co-discovered of MP470, holds patent on MP-470David Bearss, Co-discovered of MP-470 and employee of Surgen, holds patent on MP-470All other authors have no conflicts of interest.JW developed the ideas for these experiments, performed much of the work, and drafted the manuscript. DM was involved with experimental design and provided resource to perform this work. RE performed some of the experiments. LC provided technical laboratory support. DB provided resource and technical advice. BS provided resources and participated in the design and coordination. All authors read and approved the final manuscript.


H. pylori infection is less prevalent in childhood. This study validated whether the rates of H. pylori colonization depend on different acquisition ages, and correlate with the different gastric Lewis antigens or cytokine expressions after H. pylori acquisition.H. pylori and 20 mice served as naïve control. The success of H. pylori colonization was assessed on the 2nd week and the 8th week, respectively. The intensity of the Lewis x, sialylated Lewis x, and cytokine expressions, including TNF-α, IFN-γ, IL-6, IL-10, and IL-1β, were immunochemically stained and graded.We applied a young (7-day-old) C57BL/6 mice group (n = 50) and adult (6-week-old) C57BL/6 mice group (n = 50). In each group, 30 mice were challenged with nd week after H. pylori challenge, the colonization rates of H. pylori were similar between the young mice group and the adult mice group . However, on the 8th week, the H. pylori colonization rate was significantly lower in the young mice group than in the adult mice group . On the 8th week, the young mice with a persistence of H. pylori colonization had higher sialyl-Lex, higher IL-10, and lower IFN-γ than those of the mice that lost colonization during the 2nd to the 8th week (P < 0.05).On the 2H. pylori colonization could be an acquisition-age determinant process. After H. pylori exposure at an early acquisition age, the host response with a higher sialyl-Lex and IL-10, but a lower IFN-γ correlates to the consequent persistence of H. pylori colonization.The persistence of  Helicobacter pylori infection can cause chronic gastritis and even peptic ulcer disease in both children and adults [H. pylori has been shown to be less in children than in adults [H. pylori infection in childhood may possibly lead to a subsequent loss of colonization [H. pylori colonization after early acquisition remains not well-known.d adults ,2. In epn adults ,4. A foln adults . Severalnization -6. HowevH. pylori selectively binds to the fucosylated Lewis antigen (Leb) of gastric cells mediating a heavy bacterial colonization [H. pylori binds to the gastric sialylated Lewis x antigen  alternatively assisting the persistent colonization after chronic inflammation [b antigen of the host is absent or weak [H. pylori-infected children had a significant increase of sialyl-Lex, but not Leb expression [H. pylori in childhood could be related with the variable host responses of gastric inflammation or of epithelial sialylation to adapt the SabA of H. pylori.A blood group binding adhesin (BabA) of nization -9. The oammation , and esp or weak . Our recpression . It is tH. pylori strains can successfully colonize most of the mice for more than six months [H. pylori infection leads to diverse Th1 type predominant gastric inflammations with variable pro-inflammatory and Th1-dominant type cytokine responses among different hosts [H. pylori colonization could be an acquisition-age determinant process. In addition, this study attempted to answer whether the variable sialyl-Lex and the inflammatory cytokine expressions after H. pylori challenge could determine the persistent colonization of H. pylori infection, especially in mice receiving the challenge of early acquisition.With adult C57BL/6 mice older than six weeks of age, the clinical x months ,14. The nt hosts -18. AccoH. pylori clinical isolate (HP 625) and 40 in naïve controls. The multiplications of the H. pylori isolates were applied with the same method as our previous mice model study to achieve a density of 0.8 × 108 CFU/ml. [nd and the 8th week. In the experimental mice, ten each of the young and adult mice at the 2nd week and 20 each of the young and adult mice at the 8th week after inoculation were sacrificed by intra-peritoneal injection of pentobarbital (150–200 mg/kg). The isolated stomachs were incised along the greater and lesser curvatures into two halves. One half was formalin-embedded for histology and Lewis antigen examinations. The other half was immediately placed in buffer, frozen on dry ice, and stored at -80°C for cytokine examination. H. pylori infection was proven by a rapid urea test and histological examination.This study used 100 C57BL/6 mice , including 60 in the experimental group challenged with a type 1, SabA-positive  CFU/ml. . For theH. pylori density (range 0–4), acute inflammatory score , chronic inflammation score , atrophic change , and intestinal metaplasia  were evaluated topographically over the antrum, corpus and cardia, respectively. The grading system of H. pylori density used was as follows: 0, no bacteria detected; 1, a mild level of colonization and bacteria not detected in every gastric crypt; 2, a mild level of colonization with bacteria detected in the majority of crypts present; 3, moderate to heavy colonization in all crypts; and 4, severe colonization with all crypts densely packed with bacteria. The results presented are the mean value of colonization for each group. The total H. pylori density (THPD) was defined as the sum of the densities obtained from the antrum, corpus and cardia, and thus ranged from 0 to 12. The total acute (TAIS) and chronic (TCIS) inflammatory scores were also a sum of the score from three locations (range 0–9).Two investigators including one pathologist, unaware of the demographic data and colonization results, analyzed the gastric histology. Applying the method of our previous mice model , the hisH. pylori was defined as the presence of any bacteria in the gastric sections, while mice were considered to have failed to be inoculated when no bacteria were seen in histology and negative for the CLO test.Bacterial infection of x, Signet Laboratories, Inc., Dedham, MA) was selected to detect the gastric Lex antigens [x antigen staining, the monoclonal antibody against sialyl-Lex  was used [x (range 0–5) and sialyl-Lex (range 0–4) was scored according to the topography of the staining .Immunostaining of gastric specimens for Lewis antigens was performed using the avidin-biotin-peroxidase technique with a VECTOR Ms.O.M. Immunodetection Kit. The primary monoclonal antibody , and on the lamina propria components, including the matrix, polymorphonuclear cells, lymphocytes, and fibrocytes (Table The frozen sections (4 μm) were dried at room temperature for 2 h, and fixed with 3.7% formaldehyde for 1 min. After washing with PBS twice, the samples were fixed with cold acetone and then washed with PBS. The Mouse Ig Blocking Reagent  was used for blocking nonspecific binding sites. The rat anti-mouse monoclonal antibodies  and the rabbit anti-mouse polyclonal antibody (IL-1β) were used as the primary antibody. The HRP polymer conjugate broad spectrum  was used as the 22 test was used to test the difference of colonization rates between the experimental and control mice. The histological parameters, Lewis antigens, and the cytokine expressions were compared with the Fisher's exact test for different subgroups. The Friedman test was used to test the difference of H. pylori density between the antrum, corpus and cardia of the stomach in each mouse. All tests were assessed two-tailed and a p value < 0.05 was taken as significant.The Pearson's χnd week after H. pylori challenge, H. pylori colonization was successful in 89% (8/9) of the young mice group and 100% (10/10) of the adult mice group. In contrast, on the 8th week after H. pylori challenge, the colonization rate was significantly lower in the young mice than that in the adult mice . For those mice with successful colonization either on the 2nd or the 8th week, the densities of H. pylori were evenly distributed on the antrum, corpus, and cardia in both young and adult mice (P > 0.05).On the 2x and sialyl-Lex were weak on the gastric antrum. However, a gradual increase of the total intensity of Lex and sialyl-Lex expressions was disclosed in the age increments .In the early life of the mice, the positive IFN-γ staining was in the majority found on the epithelium and matrix, as the intensity of positive IFN-γ staining decreased after age ≥ 5 weeks  than the control mice , but such a marginal significance was not disclosed in the adult mice . Moreover, in the young mice group, the mice with persistent colonization on the 8th week had an evidently higher rate of high TCIS than the mice that lost colonization . On the 2nd week after H. pylori challenge, the percentage of colonized young mice having a high intensity of sialyl-Lex expression (score ≥ 7) had dropped compared to that in the control mice . However, such a drop was not evident on the 8th week  and with adult mice on the 2nd week  and the 8th week  after H. pylori challenges.On the 2nd week after H. pylori challenge, the young mice with successful colonization had a significantly higher intensity of IL-6 on fibrocytes  and matrix , but lower IFN-γ  on the matrix than the naïve controls (Table P = 0.02).On the 2th week after H. pylori challenge, the rates of high TNF-α  on the matrix and high IL-1β  on fibrocytes were higher in the young mice with persistent colonization than in the control mice (Table P = 0.003), IL-10  and IL-6  on fibrocytes were also significantly lower than those of the control naïve mice. Nevertheless, it was more common in the colonized adult mice than the control mice to have a higher IFN-γ (80% vs. 40%), IL-10 (80% vs. 40%), and IL-6 (80% vs. 30%) on the matrix.On the 8ce Table . For thend week, the colonized young mice had no significant difference in the intensity of sialyl-Lex expression than the control mice (P > 0.05). However, on the 8th week after H. pylori challenge, the young mice with persistent colonization had a higher rate of evident sialyl-Lex expression (score ≥ 7) than those of the mice that lost colonization . In addition to showing a higher rate of sialyl-Lex expression (score ≥ 7), all of the persistently H. pylori-colonized young mice significantly coexisted with lower IFN-γ and higher IL-10 expressions than those mice that had lost persistent colonization on the 8th week (Table P = 0.008).On the 2th week after H. pylori challenge. None of the young mice with persistent bacterial colonization had IFN-γ expression on the epithelium but had obvious IL-10 expression on the matrix and lymphocytes as compared to the mice without colonization.Table H. pylori mainly occurs in childhood and spontaneous elimination of infection usually occurs in young children [H. pylori colonization rate of isolates ranging to more than 80%, which is compatible with previous reports [H. pylori in adult human patients usually results in chronic colonization [nd week of young mice after H. pylori acquisition, the colonization rate in the 8th week was 53%, which supports that spontaneous elimination of H. pylori really occurs during the early acquisition age of mice. So the present study should be highly original to introduce a mouse model designed to validate that there should be a loss of H. pylori colonization after an earlier acquisition of H. pylori, which closely mimics the clinical findings in young children.Acquisition of children -6,20. In reports ,14. Thisx is positively related to the increment of age in humans [H. pylori infection, the expression of sialyl-Lex on gastric epithelium is weak or lacking in dyspeptic children and adults [x of mice is also positively correlated to the increment of age , but not on the 8th week  after H. pylori challenge. Assuming that dense inflammation causes more severe clinical symptoms, this histology change could be correlated to our previous findings that H. pylori infection could cause short-term epigastric pain in children, although usually resolved at follow-up [H. pylori acquisition in children usually presents as a self-limited sequel in inflammation.In this animal model, early ollow-up . ConcernH. pylori infection, the different host responses of the gastric cytokines could possibly be related to different clinical outcomes [After outcomes ,18,23,24outcomes . Our micnd and the 8th week after H. pylori challenge [H. pylori infection have been demonstrated to be Th1-dominant, because the level of IFN-γ is increased in H. pylori-infected subjects compared to controls [H. pylori-related gastritis, especially during chronic inflammation and potentially attributed as a protective role against H. pylori colonization [P = 0.048). This finding is compatible with a human study in children which showed that children with H. pylori infection had a lower concentration of IFN-γ than in non-infected controls [H. pylori infection outcomes in children.Based on the immunohistochemical study, this study confirmed that the pro-inflammatory cytokine expressions on the gastric lamina propria could change on both the 2hallenge ,26. Bothcontrols ,18,23,27nization -18,28. Acontrols . This daH. pylori challenge to protect the gastric mucosa [H. pylori colonization had a 100% coexistence with a high IL-10 expression on the matrix and lymphocytes. These data once again support that upregulation of IL-10 in such mice should be a co-existed compensatory regulation after a drop in IFN-γ to serve as a protective role for the infected mucosa. In addition to coexistence with high IL-10, the persistently H. pylori-colonized young mice exclusively had low IFN-γ and high sialyl-Lex co-expressions on the epithelium compared to those without colonization. These data once again suggest that the host response having a low IFN-γ should somewhat coexist with a high sialylation of Lewis antigen, which can possibly serve as putative receptors for H. pylori colonization.IL-10 is a Th2 cytokine, and can be triggered by compensatory regulation after a Th1 shift by the c mucosa ,15,23,27H. pylori represents a similar course of H. pylori infection to that in early childhood. A high expression of sialyl-Lex and a depletion of IFN-γ on the gastric epithelium coexisting with high IL-10 in infected young mice should be related to maintaining the persistent colonization of H. pylori. Targeting the sialylation or enhancing the IFN-γ expression on the gastric epithelium should be promising to decrease the persistent colonization of H. pylori in childhood or even in adulthood.In conclusion, this animal model with early acquisition of The authors guarantee that there is no financial relationship with any company. There are no competing interests including the study design, the collection, analysis, and interpretation of data, the writing of the report, and the decision to submit the paper for publication.YYJ contributed to the study design, study performance, and wrote the first draft of the manuscript. YHB, an experienced pathologist, read the histology and Lewis staining of each biopsy. WJJ also contributed to the study design, technique consultation, and paper revision. SBS, the correspondence author, organized the whole study design, team discussion, and final revision of this paper.


Dietary restriction (DR) extends the lifespan of a wide variety of species and reduces the incidence of major age-related diseases. Cell senescence has been proposed as one causal mechanism for tissue and organism ageing. We show for the first time that adult-onset, short-term DR reduced frequencies of senescent cells in the small intestinal epithelium and liver of mice, which are tissues known to accumulate increased numbers of senescent cells with advancing age. This reduction was associated with improved telomere maintenance without increased telomerase activity. We also found a decrease in cumulative oxidative stress markers in the same compartments despite absence of significant changes in steady-state oxidative stress markers at the whole tissue level. The data suggest the possibility that reduction of cell senescence may be a primary consequence of DR which in turn may explain known effects of DR such as improved mitochondrial function and reduced production of reactive oxygen species. Dietary restriction (DR), whereby total caloric intake is reduced but adequate nutrition is maintained, results in an extension of lifespan. Additionally, DR has been shown to delay the onset and severity of cancer and other diseases associated with ageing . The DR This is even more true for the response to adult-onset, short-term DR. While the effect on lifespan becomes less robust if DR is implemented in older animals ,11, therin vitro [in vivo [in vivo [Recently, evidence is mounting that cellular senescence, which was originally described as the permanent loss of replicative capacity in human fibroblasts in vitro , is a co[in vivo -23. Sene[in vivo -28.High [in vivo ,30 and w[in vivo -34. Sene[in vivo  and secr[in vivo , inflamm[in vivo ,22,37 an[in vivo ,39.If this concept were correct, one would hypothesize that a reduction of cell senescence might be part, and potentially a causal part, of the beneficial action of DR. This would be interesting because less senescent cells could explain the anti-inflammatory and anti-oxidative action of DR. However, there are few data to support such a hypothesis. There is good evidence that both life-long and adult-onset DR limit T cell senescence in mice and primates ,40-42, aINK4a and p19Arf in the kidney. However, the specificity and sensitivity of sen-β-Gal as a marker for senescent cells in vivo has been repeatedly questioned [INK4a and p19Arf expression was similarly changed in postmitotic tissues like brain cortex and heart, suggesting that expression from the INK4A locus might be a better indicator for aging than for cell senescence. Further indirect evidence for decreased cell senescence under DR came from a study showing reduced levels of IGFBP3, a major secretion product of senescent epithelial and mesenchymal cells, following long-term DR [There is very little data available on the impact of DR on cell senescence in solid mammalian tissues. Early data ,46 showeestioned ,48. More-term DR . However-term DR  and babo-term DR , no decr-term DR . To our We tested the impact of short-term (3 months), adult-onset DR on cellular senescence in mice. We concentrated on the small intestine, a highly proliferative organ, and on liver with a slow cell turnover under non-pathological conditions. We had shown before that senescent cell frequencies in these organs increase significantly during normal aging in mice . Using sMale C57/BL mice were subjected to three months of DR by average 26% of food restriction starting at 14 months of age. The study cohort is characterized in 2=0.7080). γ-H2A.X staining on its own may overestimate frequencies of senescent cells, especially in tissue compartments with high proliferative activity such as gut because an active DNA damage response can also be initiated by replication stress in dividing cells. Accordingly, we showed recently that a combination of strong positivity for γ-H2A.X with absence of a proliferation marker results in quantitatively correct estimates of senescent cell frequencies in vitro and in vivo[We first measured the frequency of intestinal enterocytes showing an active DNA damage response as characterized by nuclear positivity for the DNA damage marker, γ-H2A.X. As we have shown before, there were few γ-H2A.X-positive enterocytes within villi, instead, positive cells centered around the transient amplifying zone in crypts . DR signd in vivo. Double d in vivo. FrequenIn liver, frequencies of γ-H2A.X positive hepatocytes were higher in centrilobular than periportal areas Figure  as shownDespite the presence of active telomerase, telomeres shorten with age in various tissues of laboratory mice ,53. HoweSenescent cells are a major source of ROS because mitochondrial dysfunction and, possibly, other ROS-producing mechanisms are part of the senescent phenotype ,55-57. L4-HNE is a major end product of lipid peroxidation and has been shown to accumulate in tissues with age . We founBroad-band autofluorescence originates mainly from oxidised and cross-linked cell components, like advanced glycation end products (AGEs) and lipofuscin and is thus regarded as a good cumulative marker for oxidative damage , 60-62. 2O2 release rate from tissue homogenate are indicative of steady-state levels of oxidative stress/oxidative damage. These markers were measured in whole liver homogenates. None of them were significantly different between AL and DR mice , nitrotyrosine content (a marker for oxidative protein damage) and He Figure . SimilarThis is the first study to show that short-term DR reduced frequencies of senescent cells in solid tissues. It is important to note that the magnitude of the reductions, amounting to between 3.3 and 6.5% depending on the tissue compartment, is very substantial given the short duration of the treatment. Frequencies of senescent cells increase with age in intestinal crypts and liver at rates below 0.5% per month , indicatin vitro is associated with a 3- to 5-fold increase in cellular ROS levels [2O2 release as acute parameters are less sensitive than the cumulative markers mentioned above. Moreover, an average decrease in the number of senescent cells by about 5% in liver would result in less than 10% decrease in total ROS, which is within the experimental error for these measurements. As there are very few senescent cells in villi, the expected impact of the observed decreases in senescent cell frequencies in the crypts on ROS in the whole intestinal mucosa would be even lower.Cell senescence S levels ,55-57. VS levels ,56,65. AS levels ,59, 62. in vitro[Taken together, our data suggest that at least some of the beneficial effects of DR that have been repeatedly described in the literature, such as improved mitochondrial coupling and reduced ROS release ,58, coulin vitro. The samin vitro,32.Our results lead to the question of how DR could impact directly on cell senescence. One interesting candidate may be signaling through the mTOR-S6K1 pathway. DR reduced phosphorylation and activity of Akt1, mTOR and its downstream targets S6K1 and 4E-BP1 . KnockouIn conclusion, the data are compatible with the idea that reduction of cellular senescence is a primary effect of DR, possibly mediated via suppression of signaling through mTOR-S6K1, and that this reduction in turn might be sufficient to account for the improvement of mitochondrial function and reduction of ROS production that are known to occur under DR.2. The intestinal mucosa was stripped from the muscle layer before freezing. Further details of the experimental protocol can be found as From a group of 90 male C57/BL mice aged 14.2 ± 1.2 months, 45 animals were subjected to DR, while the other 45 animals, matched for body mass, food intake and age, served as ad libitum-fed (AL) controls. The experiment lasted for 3 months with an average food restriction of 26%. All mice were sacrificed at the end of the experiment. Five mice per group were perfused by whole animal fixation with 4% paraformaldeyde followed by dissection. Tissues were paraffin-embedded and 5μm sections were prepared from small intestine and liver. Tissues from five additional mice per group were frozen in OTC for cryosectioning. Tissues from further animals were frozen in liquid NSen-β-Gal histo-chemistry, immunohistochemistry and telomere Q-FISH were performed as described . The antFor double immunofluorescence, blocked sections were incubated with anti-PCNA and anti- γ-H2A.X anti-bodies together in PBS at 4°C overnight and incubated with Alexa-555-conjugated goat anti-rabbit antibody and biotinylated anti-mouse antibody for 45 min in PBS. Subsequently, tissue sections were washed 3 times and incubated with 0.2% Fluorescein Avidin-DCS in PBS for 30min. Images were taken in a Leica DM5500B microscope with 40x objective. 30-40 crypts were scored for each animal.Telomerase activity was measured using the TeloTAGGG Telomerase PCR ELISA kit (Roche) according to the manufacturer's recommendations.Autofluorescence was measured on unstained, non-deparaffinized tissue sections using a Leica DM5500B microscope. The sample was excited at 458nm and fluorescence emission captured above 475nm.TM LC-18S column (250 X 4.6 mm)  and an electrochemical detector . The mobile phase consisted of 10% aqueous methanol containing 94 mM KH2PO4, 13 mMK2HPO4, 26 mM NaCl and 0.5 mM EDTA. Elution was performed at a flow rate of 1.0 ml/min with a lower absolute detection limit of 40 fmol for 8-oxo-dG, or 1.5 residues/106 2'-deoxyguanosine (dG). dG was simultaneously monitored at 260 nm.The base oxidation product 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) was detected by HPLC with electrochemical detection (ECD). Ground frozen tissues  were thawed and genomic DNA was obtained using standard phenol extraction . The DNATM protein assay kit . Nitrotyrosine was detected by oxiSelectTM Nitrotyrosine ELISA kit  according to the protocol provided by the manufacturer.Ground frozen tissues  were thawed and total protein was extracted using Microplate BCAGround frozen tissue was homogenized in PBS and used immediately for the assay. The rate of hydrogen peroxide release was monitored fluoro-metrically as resorufin formation due to oxidation of Amplex Red  in the presence of horseradish peroxidase (2U/ml), at an excitation 544 nm and an emission 590 nm using a FLUOstar Omega (BMG Labtech). Superoxide dismutase (75U/ml) was included in the assay buffer. The slope was converted into the rate of hydrogen peroxide release with a standard curve. Protein concentration was measured using Bio-Rad DC protein assay kit.Table S1.


With the availability of high-throughput microarray technologies, investigators can simultaneously measure the expression levels of many thousands of genes in a short period. Although there are rich statistical methods for analyzing microarray data in the literature, limited work has been done in mapping expression quantitative trait loci (eQTL) that influence the variation in levels of gene expression. Most existing eQTL mapping methods assume that the expression phenotypes follow a normal distribution and violation of the normality assumption may lead to inflated type I error and reduced power. QTL analysis of expression data involves the mapping of many expression phenotypes at thousands or hundreds of thousands of marker loci across the whole genome. An appropriate procedure to adjust for multiple testing is essential for guarding against an abundance of false positive results. In this study, we applied a semiparametric quantitative trait loci (SQTL) mapping method to human gene expression data. The SQTL mapping method is rank-based and therefore robust to non-normality and outliers. Furthermore, we apply an efficient Monte Carlo procedure to account for multiple testing and assess the genome-wide significance level. Particularly, we apply the SQTL mapping method and the Monte-Carlo approach to the gene expression data provided by Genetic Analysis Workshop 15. With the availability of high-throughput microarray technologies to measure the expression levels of many thousands of genes simultaneously, investigators have developed a vast amount of statistical and computational methods for analyzing microarray data in the last decade. On the other hand, because of the abundance of single-nucleotide polymorphisms (SNPs) as well as the modern genotyping technologies, tremendous efforts have been focused on the genetic mapping of complex human diseases, many of which are associated with quantitative traits. However, limited work has been done in combining gene expression data and marker genotype data and detecting expression quantitative trait loci that influence the variation in levels of gene expression.There are important challenges in mapping of eQTL. First, it is well known that microarray data are noisy due to systematic biases and appropriate normalization procedures are needed to adjust for such biases. Many expression phenotypes may be non-normally distributed even after proper normalization procedures, as is evident by the gene expression data provided by Genetic Analysis Workshop 15 (GAW15). Commonly used eQTL mapping methods such as the standard variance-component (VC) approach implemented in programs SOLAR  assume tTo overcome the aforementioned challenges, we first applied the semiparametric quantitative trait loci mapping method of Diao and Lin  to gene Very recently, Diao and Lin  proposedH0 : HA : i, j)th component corresponding to the result for mapping the ith expression phenotype at the jth marker on the genome, where For the gene expression data, we repeatedly applied the SQTL and perform a genome-wide linkage scan for each expression phenotype. The resultant likelihood ratio test statistics or LOD scores for testing In eQTL mapping, one performs testing on thousands of marker loci for each expression trait and one thus needs to adjust for multiple testing to guard against an abundance of false-positive results. Carlborg et al.  suggestep-values as described in Lin [To apply the Monte Carlo approach to the gene expression data, we assess the genome-wide significance level for each expression trait and obtain the trait-specific adjusted d in Lin . Note thd in Lin .p-values for each trait were based on 100,000 Monte Carlo samples. The average thresholds  at the genome-wide significance levels of 0.05, 0.01, 1.0 × 10-3, 1.0 × 10-4, and 1.0 × 10-5 are 1.34, 1.70, 2.50, 3.59, and 4.68 corresponding to point-wise p-values of 6.5 × 10-3, 2.6 × 10-3, 3.5 × 10-4, 2.4 × 10-5, and 1.7 × 10-6, respectively. The relatively low thresholds compared to those used in Morley et al. [The gene expression data provided to GAW15 include 14 three-generation CEPH (Centre d'Etude du Polymorphisme Humain) Utah families, each of which consists of 13 or 14 individuals. We performed genome-wide linkage scans for expression levels of 3554 genes at 2882 SNPs using SQTL. We utilized SOLAR to estimate the identity-by-descent (IBD) allele-sharing probabilities at each SNP locus after eliminating Mendelian inconsistencies. Gender is included in the model as a covariate. The genome-wide significance levels and the adjusted -3, 1.0 × 10-4 and 1.0 × 10-5, respectively. We detected 58 more expression traits at the genome-wide significance level of 1.0 × 10-3 than Morley et al. [At the genome-wide significance level of 0.05, for 954 expression phenotypes, there was evidence of linkage to specific chromosomal regions. We found 356, 200, 185, and 179 expression phenotypes with evidence of linkage at the genome-wide significance levels of 0.01, 1.0 × 10-5. At the genome-wide significance level of 1.0 × 10-3, we found 68 hotspots with six or more hits, including the only 2 hotspots on chromosomes 14 and 20, with six and seven hits each detected by Morley et al. [-5, regulators for 51 phenotypes were found to map to the window on chromosome 10 (10p12).As in Morley et al. , we divip-values of Shapiro test less than 1.0 × 10-12: 211518_s_at, 204695_at, 202982_s_at, and 202950_at. The histograms of these four traits are shown in Figure Among the total 3554 expression phenotypes, almost half of them appear to be non-normally distributed by using the Shapiro test at a significance level of 0.05. At a significance level of 0.0001, the Shapiro tests for normality are still significant for more than 19% of phenotypes. Table We further performed multipoint genome-wide linkage analysis for the four non-normally distributed phenotypes and compared the performance of SQTL with that of the standard VC approach assuming normality. The multipoint IBD probabilities were estimated by using SOLAR. The whole genome linkage scan results are shown in Figure In this work, we applied the robust and powerful SQTL method to gene expression data and applied an efficient Monte Carlo procedure to account for multiple testing and to assess the genome-wide significance level. With the application to the GAW15 data, we detected many more eQTLs and hotspots at stringent genome-wide significance levels than Morley et al. . Our newThe Monte-Carlo approach described in this work adjusts for multiple testing across the genome. It would be desirable to develop an efficient procedure to account for multiplicities across the genome and multiplicities across gene expressions simultaneously. Furthermore, the Monte-Carlo approach requires a large sample size in order to accurately approximate the joint distribution of the test statistics. Previous simulation studies demonstrated that the Monte-Carlo approach performed well for sample size as small as 50 pedigrees (results not shown). Future research is needed in evaluating the performance of the Monte-Carlo approach in small sample size situations.With the availability of the abundance of SNPs, it is desirable to perform genome-wide association analysis of expression quantitative traits following genome-wide linkage analysis. We are currently extending the semiparametric quantitative transmission-disequilibrium test  to the gThe author(s) declare that they have no competing interests.


The risk of premature cardiovascular disease in patients with familial hypercholesterolemia (FH) can be profoundly reduced by cholesterol-lowering therapy, and current guidelines for FH advocate ambitious low-density lipoprotein cholesterol (LDL-C) goals. In the present study, we determined whether these goals are reflected in current clinical practice once FH has been diagnosed.In 2008, we sent questionnaires to all subjects (aged 18–65 years) who were molecularly diagnosed with FH in the year 2006 through the screening program in the Netherlands. Of these 1062 subjects, 781 completed the questionnaire : 4.1±1.3 mmol/L). The number of persons that used cholesterol-lowering therapy increased from 397 (51%) at baseline to 636 (81%) after diagnosis. Mean treated LDL-C levels decreased significantly to 3.2±1.1 mmol/L two years after diagnosis. Only 22% achieved the LDL-C target level of ≤2.5 mmol/L.The proportion of patients using cholesterol-lowering medication was significantly increased after FH diagnosis through genetic cascade screening. The attained LDL-C levels were lower than those reported in previous surveys on medication use in FH, which could reflect the effect of more stringent lipid target levels. However, only a minority of the medication users reached the LDL-C target. Familial hypercholesterolemia (FH) is an inherited disorder of lipid metabolism that predisposes to severe premature cardiovascular disease (CVD).Although FH is relatively common (1∶400 in The Netherlands), many patients are not diagnosed at all or they are identified only after symptomatic onset of CVD.In 2001, we demonstrated that 38% of the participants used cholesterol-lowering therapy before the screening compared to 86% two years after the molecular diagnosis.In the present study, we therefore assessed whether the molecular screening program has improved the preventive care for FH. In addition, we analyzed the determinants that predict the decisions about cholesterol-lowering medication once a molecular diagnosis of FH is made.We recruited subjects from the database of the nation-wide cascade screening program for FH in the Netherlands.Subjects between 18 and 65 years of age were potentially eligible if they were visited in 2006 by a genetic fieldworker, a functional FH mutation was identified, and the lipid profile was assessed. We excluded patients if their address information was incorrect or unavailable or if they had declared that they did not want to be approached for scientific research. All probands with whom family screening was initiated were excluded as well.We sent a questionnaire by surface mail to all selected FH patients in May 2008. Two months after the questionnaire had been distributed, we sent a reminder to the subjects who had not returned the questionnaire. If we had not received the questionnaire after six months, we contacted a random sample of the non-responder population by telephone in order to elucidate the medication use at follow-up, and inquired after their reasons for non-response. We selected these non-responders per month in which they were diagnosed, and we choose the months January, April, July, and October in 2006. We subsequently phoned until 25 consecutively diagnosed individuals in each period had consented to participate, which resulted in an additional number of 100 participants.Data on demographics, CVD risk factors, lipid profile and medication use at baseline were extracted from the database of the screening organization.The primary endpoints were attained LDL-C levels and the difference of the proportions treated persons at molecular diagnosis (‘at baseline’) and on average two years after the molecular diagnosis (‘at follow-up’). The secondary endpoints were the differences in CVD risk factors and lipid profiles between the individuals who were treated or had been treated with cholesterol-lowering drugs and those who were never treated.table S1). Potency scores for statins were derived from Walma et al.When untreated LDL-C levels or recent on-treatment levels were not available, we imputed these LDL-C levels by means of the estimated LDL-C lowering potency of a specific lipid-lowering drug and dose. These potency scores were summarized in a supplementary table  in the SAS procedure GENMOD accounting for correlations within families. The association between initiation of treatment since molecular diagnosis and demographics, clinical characteristics, and lipids, was analyzed with logistic regression (GEE) for all individuals who were treatment naïve at baseline. We assessed the relation between treatment potency of the drug regimen used after the molecular diagnosis and demographic, clinical, and lipid characteristics by linear regression analyses (GEE) for all persons that had been treated at baseline and/or during follow-up. We used paired t-tests to compare LDL-C levels and potency of prescribed medication within persons over time, e.g. at baseline and after on average two years of follow-up. Variables with a skewed distribution were log-transformed before analysis. A p-value<0.05 was considered significant. Statistical analyses were performed with the SAS package version 9.1 .During 2006, a pathogenic LDLR or apolipoprotein B (ApoB) mutation was identified in 341 probands with a clinical diagnosis of FH. In the same period, a total of 4228 relatives aged between 18 and 65 years were screened for the presence of the specific mutation that was identified in the proband. Such a mutation was identified in 1328 of these relatives. Of these mutation carriers, 458 (34%) were first, 257 (19%) were second, 276 (21%) were third and 312 (23%) were fourth or further degree relatives of the proband, and for 25 (2%) we could not retrieve the distance from the proband.We excluded 266 patients with FH: 134 had not given written informed consent for additional studies at the time of molecular diagnosis and of 132 we lacked up-to-date contact information. Of the remaining 1062 persons, 781 (74%) completed the questionnaire of whom 100 completed it upon a telephone call. The two main reasons for not returning the questionnaire by surface mail were that they had not received the two mailings (n = 33) or that they had been too busy (n = 30).The 781 participants had a mean age (± standard deviation (SD)) of 42±12 years and a mean LDL-C level at diagnosis of 4.1±1.3 mmol/L and 359 (46%) were male table 1.The 281 non-participants, i.e. those who did not respond to the questionnaires and could not be reached by telephone, were significantly younger , had a lower prevalence of CVD , were more often smokers  and used less cholesterol-lowering medication at baseline  than the 781 participants.At baseline, 397 of the 781 subjects (51%) already used cholesterol-lowering medication and 239 of 384 initially untreated persons (62%) started cholesterol-lowering treatment during follow-up figure 1Nearly one fifth (n = 145) of the 781 participants never started medication. The main reasons were physicians' advice ; own choice ; intention to become pregnant, pregnancy or to breastfeed .After two years of follow-up, the mean LDL-C level (± SD [interquartile range (IQR)]) of all participants, was significantly reduced by 22% as compared with the level at baseline . In the group of 239 treatment naïve persons that started cholesterol-lowering medication after diagnosis the mean LDL-C level (± SD) was decreased with 44% from 5.1±1.3 at baseline to 2.8±0.80 mmol/L (p<0.001) two years later. The mean additional reduction in patients who were already treated at baseline was 8%: from 3.5±0.9 to 3.2±0.9 mmol/L (p<0.001).The mean baseline LDL-C levels ± SD [IQR] differed significantly between 239 treatment naïve subjects at diagnosis who started medication during follow-up and 145 subjects who did not initiate treatment . The mean age and sex specific percentiles of the untreated LDL-C levels were considerably higher in the subjects that initiated medication after the molecular diagnosis than those who did not .The 636 persons, who already used medication at baseline or started during follow-up (i.e. ‘treated’), were older  than those 145 persons, who had never used medication (i.e. ‘untreated’) see table 1.The proportion of ApoB mutation carriers among treated subjects did not differ significantly from the proportion among untreated subjects at follow-up . The four most prevalent LDLR mutations that had been reported to result in a severe LDL-C and CVD phenotype, i.e. V408M (exon 9), 1359-1 (intron 9), 313+1/2 (intron 3) and W23X (exon 2), were more prevalent in the treated than in the untreated group .Treated patients (n = 581) had more frequently been referred to a specialist than untreated patients: 312 (54%) visited an internist and/or a cardiologist, whereas 26 of the 145 untreated patients (18%) had visited a specialist. In fact, 65 (45%) of the untreated patients responded that they did not visit a doctor at all with regard to FH treatment.Among the 384 untreated subjects at baseline, increased age, body mass index (BMI), LDL-C and triglycerides levels and low levels of high-density lipoprotein cholesterol (HDL-C) were associated with the start of medication after molecular diagnosis in univariate analyses. After stepwise multivariate regression analysis, age, LDL-C, and HDL-C independently predicted the start of medication table 2.The mean potency of the medication of the 636 treated patients was 1.90±0.35. For the 397 subjects that already used cholesterol-lowering medication at baseline, the LDL-C lowering capacity was increased after the identification of their mutation from 1.84±0.34 to 1.99±0.36 (p<0. 001). A total of 265 (34%) participants used dairy products enriched with sterols and stanols, with a similar proportion in those did or did not use cholesterol-lowering medication .For the 636 persons that used cholesterol-lowering medication during follow-up, a number of variables were significantly associated in the univariate analyses with the potency of the drug regimen after diagnosis: increased age and BMI, history of CVD, presence of diabetes or hypertension, smoking status at diagnosis, high pre-treatment LDL-C and triglycerides, and low HDL-C. After stepwise multivariate regression analysis, untreated LDL-C levels, age, history of CVD, and diabetes were independent predictors of the potency of the drug regimen used after the molecular diagnosis table 3.Of the 781 participants, 297 (38%) persons could report a LDL-C level at the end of follow-up: the target level of ≤2.5 mmol/L was achieved in 65 (22%) persons. Based on the mean potency of the current medication (1.90), we expected that 25% of the 636 treated patients would attain the target LDL-C level. In total, 492 of the 636 (77%) subjects that used cholesterol-lowering medication after diagnosis did not achieve LDL-C levels ≤2.5 mmol/L. Of these, 176 (36%) did achieve a reduction of 50% or more in LDL-C levels. Of the 145 subjects that remained untreated, 15 (10%) already had LDL-C levels ≤2.5 mmol/L at screening.th percentile, compared to the treated persons, whose levels were at the age and sex specific 92nd percentile before initiation of cholesterol-lowering drugs.In the current study, we found that mean LDL-C levels of individuals, who were untreated at baseline, decreased by 44% with treatment in the two years after molecular diagnosis. This means that cascade screening by molecular diagnosis not only leads to identification of FH but also supports the decision to treat these new FH patients. The decision whether or not to initiate lipid-lowering treatment was found to be dependent on age, and HDL-C and LDL-C levels. The proportion of individuals on cholesterol-lowering medication increased from 51% to 81% during follow-up. In contrast, one fifth of the identified FH subjects never started cholesterol-lowering medication. In the majority of these cases the general practitioner advised to refrain from medication because their untreated LDL-C levels were not so severely increased, i.e. on average at the 68The achieved LDL-C level in all participants two years after molecular diagnosis was significantly lower than the achieved level in a similar survey in 2001: 3.2 mmol/L in 2008 vs. 4.2 mmol/L in 2001.Surveys with a similar topic have also been performed in Norway and the United Kingdom. The survey in Norway focussed on medication use after molecular FH diagnosis among first degree relatives of probands.Audits on management of FH patients in outpatient clinics specifically were recently performed in the United Kingdom (UK) and the Netherlands. In the UK, Hadfield and colleagues showed that the proportion of patients on cholesterol-lowering medication ranged between of 88-94% with on-treatment LDL-C levels between 3.0 and 3.7 mmol/L.In general, management of FH patients is not successful when measured against the new guideline targets. In fact, a sizable subset of persons with FH does not get pharmacological treatment at all and the treatment target of LDL-C levels ≤2.5 mmol/L is achieved only in 22% of the treated patients.One fifth of individuals with a molecular diagnosis of FH did not use cholesterol-lowering medication at all. Almost half of the untreated patients had not consulted a physician, which is unexpected, since genetic field workers encouraged all subjects who were found to have dyslipidemia at screening to visit their physician. Moreover, those with confirmed molecular diagnosis were explicitly urged to seek for medical consultation. The other half did not start treatment even though they had consulted a physician, which is remarkable. However, the FH patients who remained untreated had on average few cardiovascular risk factors and above all, absence of severe dyslipidemia. Whether this group of FH ‘patients’ should receive medication based on mutation carriership or not, is of scientific and clinical importance, and should be further elucidated.It is intriguing to observe that a relatively large group of subjects had a molecular diagnosis of FH but lacked a severe dyslipidemia phenotype. One of the explanations could be a high prevalence in this group of mild mutations such as ApoB mutations, which in general result in a less severe FH phenotype than LDLR mutations.One drawback of our approach is the possible bias inflicted by non-participation. Based on the characteristics of the 281 patients who did not participate, i.e. lower proportion of cholesterol-lowering medication use at baseline, younger age and lower prevalence of CVD as compared to the 781 participants, one could reason that the proportion of treated subjects at follow-up will be an overestimation. However, LDL-C levels and the proportion of subjects that used of cholesterol-lowering medication at follow-up did not differ between the 681 participants that returned the questionnaire by surface mail and the 100 subjects that completed the questionnaire by telephone. Assuming that these 100 participants by telephone contact are a random sample of the source population of which the 281 non-participants are a part, we expect the impact of potential selection bias to be modest.In conclusion, the molecular diagnosis of FH leads to an increased proportion of patients that start or intensify cholesterol-lowering medication, and consequently, to a robust decrease in LDL-C levels. The attained LDL-C levels are lower than those reported in a previous survey which could reflect the effect of more stringent lipid target levels. However, only a minority of the patients was treated with a potent drug regimen to reach set targets.Table S1Potency scoring for several cholesterol-lowering drugs. *Potency scores for statins were derived from Walma Ned Tijdschr Geneeskd 2006;150:18–23, who based the scores themselves on Law BMJ 2003;326:1423-7. #Correction factor for ezetimibe was based on 17% additional decrease in LDL-C (100%/(100%-17%) = 1.20) when added to statins . Our study population used relative low doses of bile acid sequestrants and these were estimated to have only a modest effect on LDL-C levels. Even lower potency scores were applied for fibrates and nicotinic acid, which influence primarily triglyceride and HDL-cholesterol levels respectively and have a modest effect on LDL-C levels .(0.05 MB DOC)Click here for additional data file.


Nutritional control of gene regulation guides the transformation of smooth muscle cells (SMC) into foam cells in atherosclerosis. Oxidative stress has been reported in areas of lipid accumulation, activating proliferation genes. Suppression of oxidative stress by antioxidant administration reduces this activation and the progression of lesions. We hypothesized that fish oil consumption may protect against atherosclerotic vascular disease. The study objective was to determine the effects of dietary cholesterol and fish-oil intake on the apoptotic pathways induced by 25-hydroxycholesterol (25-HC) in SMC cultures.in vivo/in vitro cell model was used, culturing SMC isolated from chicks exposed to an atherogenic cholesterol-rich diet with 5% of cholesterol (SMC-Ch) alone or followed by an anti-atherogenic fish oil-rich diet with 10% of menhaden oil (SMC-Ch-FO) and from chicks on standard diet (SMC-C). Cells were exposed to 25-HC, studying apoptosis levels by flow cytometry (Annexin V) and expressions of caspase-3, c-myc, and p53 genes by quantitative real-time reverse transcriptase-polymerase chain reaction. Results: Exposure to 25-HC produced apoptosis in all three SMC cultures, which was mediated by increases in caspase-3, c-myc, and p53 gene expression. Changes were more marked in SMC-Ch than in SMC-C, indicating that dietary cholesterol makes SMC more susceptible to 25-HC-mediated apoptosis. Expression of p53 gene was elevated in SMC-Ch-FO. This supports the proposition that endogenous levels of p53 protect SMC against apoptosis and possibly against the development of atherosclerosis. Fish oil attenuated the increase in c-myc levels observed in SMC-C and SMC-Ch, possibly through its influence on the expression of antioxidant genes.An Replacement of a cholesterol-rich diet with a fish oil-rich diet produces some reversal of the cholesterol-induced changes, increasing the resistance of SMC to apoptosis. Gene expression is influenced by dietary fat , gene trvia plasma lipids [In the 1970 s, a very low incidence of death from ischemic heart disease was observed in Greenland Eskimos, who have a high fish consumption . Evidenca lipids  to drasta lipids .via apoptosis [However, the effects of fish oil on the regulation of apoptosis are less well known . It has poptosis , destabipoptosis , which ipoptosis . In athepoptosis .via non-transcriptional mechanisms. The cellular response to p53 depends on the cell type and p53 expression level and on the presence of other apoptotic stimuli. Thus, low-level p53 expression often induces growth arrest, whereas apoptosis is only induced by higher expression levels. The induction of growth arrest may also reduce the sensitivity of cells to apoptosis [25-hydroxycholesterol (25-HC) induced apoptosis in a leukemia model system , and 25-poptosis . In humapoptosis . Endogenpoptosis . It was poptosis .in vitro study of the transformation into foam cells of SMC induced in vivo by cholesterol diet, isolating the cells before plaque formation [Our group has developed an experimental culture model of SMC isolated from aorta of cholesterol-fed chicks (Ch-SMC) and control chicks on a standard diet (C-SMC) cultured under the same conditions. It has proven suitable for ormation . Previouormation ,32. In aormation . Hence, ormation .in vivo/in vitro SMC culture model of chick experimental hypercholesterolemia and that fish oil supplementation after cholesterol dietary could change these results. We previously characterized 25OHC-induced SMC apoptosis by studying antiapoptotic and proapoptotic  gene expression [The hypothesis of this study was that vascular SMC apoptosis gene expression may be affected during early atherosclerosis in the pression . The objad libitum.The protocol of this study was approved by the Animal Laboratory Service of the University of Granada (Spain), and the chicks received humane treatment according to the regulations for Animal Research of the European Union. Newborn White Leghorn male chicks , supplied by the Animal Laboratory Service of the University of Granada, were kept in a chamber with a light cycle from 9 am to 9 pm and controlled temperature of 29-31°C with food and water available The diet was started at hatching and maintained until the chicks were killed at 20 days. Water was always available. None of the chicks died a natural death during the treatment or developed any disease. Three groups of 20-day-old chicks were used: control diet group (C-group), kept on a standard diet (Sanders A-00); Ch-treatment group (Ch-group), fed on the same diet supplemented with 5% w/w powdered cholesterol mixed homogeneously ; and a Ch-fish oil group (Ch-FO-group), fed for 10 days with the 5% cholesterol-supplemented diet and then for a further 10 days with the standard diet supplemented with 10% fish (menhaden) oil. Experimental diets were prepared daily to minimize oxidation. The standard diet contained (w/w) 42% carbohydrate (mainly starch), 3.5% fat and 20.5% protein. The fatty acid composition of the diets is given in Table 2). Media were renewed three times a week. Secondary cultures were initiated after either low or high passages using 0.05%/0.02% Trypsin-EDTA solution. All experiments were conducted using 3 or 5 passages. Cells were identified as vascular SMC by their hill-and-valley configuration at confluence and positive fluorescence staining for smooth muscle actin and myosin.SMC were isolated from the aortic arch of the chicks as described elsewhere with slight modifications  and cultCytotoxicity was analyzed by using the MTT assay -2,5-diphenyltetrazolium, bromide; Sigma). SMC were plated in 96-well plates at a density of 25 000 cells/well. After adhering during overnight culture, cells were treated for 24 and 48 h with 25-HC (5-40 μg/ml) dissolved in absolute ethanol. The final concentration of ethanol in the culture medium never exceeded 0.8% and no effect on culture was observed at or below this concentration.MTT was dissolved in DMEM at a concentration of 5 mg/ml. An amount of this solution equal to 10% of the culture medium volume was added to cell cultures. After 2 h, cultures were removed from the incubator and washed with PBS. The formazan crystals were solubilized by adding 200 μl of solubilization solution (0.05 N HCl in isopropanol). Metabolic activity was quantified by subtracting light absorbance at 630 nm from absorbance at 570 nm.2, 1 mM MgCl2, 4%BSA) to a density of 1 × 106 cells/ml with annexin V-fluorescein isothiocyanate (FITC) (0.5 μg/ml). Cells were gently mixed and incubated for 15 min at room temperature in the dark. They were then re-suspended in 1x binding buffer, and propidium iodide (PI) (0.6 μg/mL) at 4°C was added to each tube before analysis. Fluorescence was induced with the 488-nm argon laser and monitored at 512 nm (FL1) for the FITC signal and 620 nm (FL2) for IP fluorescence on FACS Vantage . The log of annexin V-FITC fluorescence was plotted against the log of IP fluorescence.Apoptotic cell death in SMC was assessed by flow cytometry . The culture medium of each plate (containing cells detached during the cell death process) was recovered in a tube, and the cells were suspended by a brief trypsinization (0.05% with EDTA) and washed twice with cold PBS. Cells were then resuspended in 100 μL of 1x binding buffer . Real-time PCR was performed with the Fast Start DNA Master SYBR Green I Kit (Roche) and Light Cycler system (Roche). For the Light Cycler reaction, a master mix of the following reaction components was prepared to the indicated final concentration: 12.6 μL H2O, 2.4 μl MgCl2 (4 mM), 1 μL forward primer (0.5 μM), 1 μL reverse primer (0.5 μM), and 2.0 μL of the Fast Start DNA Master SYBR Green I mix (Roche). The primer sequences used in this study are listed in Table in vivo and in vitro) were diluted 1:100 and amplified to obtain the Cp value for each sample. Light Cycler products of the different gene expressions were analyzed by agarose gel electrophoresis, and a Light Cycler melting curve was constructed to test for a single product at the end of each PCR reaction. A mathematical model developed by Pfaffl [Total RNA was isolated with Tri-Reagent/Trizol . Single-stranded cDNA was synthesized from 4 μg total RNA using an Oligo(dT)y Pfaffl  was usedt-test or, when the variances of two datasets significantly differed, by Welch's alternate t-test InStat statistical package was used for the data analyses. A p-value ≤ 0.05 was considered significant.Results are expressed as mean ± SEM. Data were analyzed by Student's in vivo treatments (baseline) and after in vitro treatments. Figure The percentage apoptosis was quantified in the three types of SMC cultures  after Cells showed a marked increase in apoptosis after the addition of 25-HC (p < 0.001), with a higher increase in SMC-Ch than in SMC-C (p < 0.01).At baseline, no significant difference in c-myc expression Figure  was obseAddition of 25-HC to the cultures produced a significant increase in c-myc expression (p < 0.001), with a significantly lower increase in SMC-Ch-FO versus SMC-C (p < 0.01) Figure . FinallyThe dedifferentiation and proliferation/apoptosis of SMC in the arterial intima are among changes in early atherosclerotic lesions, when the disease is still reversible ,38. The SMC from chicks fed for 20 days with a cholesterol-rich diet (SMC-Ch) were more susceptible to apoptosis than those from control chicks on a standard diet (SMC-C). This may be due to the accumulation of lipids in SMC, since lipid accumulation in early atherosclerosis has been associated with cell proliferation ,42. In cThe expression of p53 gene was elevated in SMC-Ch-FO, supporting the proposition that endogenous levels of p53 protect SMC against apoptosis and possibly against the development of atherosclerosis . The maiIt has been reported that c-myc expression interferes with the control of SMC proliferation , and culThe significant increase in p53 mRNA in the three culture types is consistent with findings that ox-LDLs  induce p53 . These rconclusion, 25-hydroxycholesterol-induced apoptosis of SMC is mediated by a significant increase in c-myc and p53 levels. These changes were generally more marked in SMC-Ch than in SMC-C, indicating that dietary cholesterol produces changes in SMC that make them more susceptible to 25-hydroxycholesterol-mediated apoptosis. Replacement of a cholesterol-rich diet with a fish oil-rich diet produces some reversal of the cholesterol-induced changes, increasing the resistance of SMC to apoptosis.In The authors declare that they have no competing interests.All authors participated in the design of these studies and carried out the different assays. A.L. drafted the manuscript. All authors read and approved the final manuscript.


There has been a long standing interest in the identification of medicinal plants and derived natural products for developing cancer therapeutics. Our study focuses upon pancreatic cancer, due to its high mortality rate, that is attributed in part to the lack of an effective chemotherapeutic agent. Previous reports on the use of medicinal plant extracts either alone or alongside conventional anticancer agents in the treatment of this cancer have shown promising results. This work aims to investigate the therapeutic properties of a library of medicinal plants from Bangladesh.56 extracts of 44 unique medicinal plants were studied. The extracts were screened for cytotoxicity against the pancreatic adenocarcinoma cell line Panc-1, using a label-free biosensor assay. The top cytotoxic extracts identified in this screen were tested on two additional pancreatic cancer cell lines (Mia-Paca2 and Capan-1) and a fibroblast cell line (Hs68) using an MTT proliferation assay. Finally, one of the most promising extracts was studied using a caspase-3 colorimetric assay to identify induction of apoptosis.Petunia punctata, Alternanthera sessilis, and Amoora chittagonga showed cytotoxicity to three cancer cell lines with IC50 values ranging between 20.3 - 31.4 μg/mL, 13.08 - 34.9 μg/mL, and 42.8 - 49.8 μg/mL, respectively. Furthermore, treatment of Panc-1 cells with Petunia punctata was shown to increase caspase-3 activity, indicating that the observed cytotoxicity was mediated via apoptosis. Only Amoora chittagonga showed low cytotoxicity to fibroblast cells with an IC50 value > 100 μg/mL.Crude extracts of Based upon the initial screening work reported here, further studies aimed at the identification of active components of these three extracts and the elucidation of their mechanisms as cancer therapeutics are warranted. Pancreatic cancer is the fourth leading cause of cancer-related death in both sexes in the United States . AlthougThis study provides data on the cytotoxic potential of 56 extracts derived from 44 different plants used in Bangladeshi folk medicine. A three-tiered screening system was designed, in which all extracts were first screened for their ability to induce death in the Panc-1 cell line using a label-free photonic crystal (PC) biosensor assay. These experiments generated biosensor images of attached cells which were used to quantify cell proliferation changes in treated versus untreated cultures. Next, extracts that showed significant cytotoxicity to Panc-1 cells (> 80% cell death at a testing concentration of 100 μg/mL) in the PC biosensor assay were tested using a colorimetric MTT assay on two additional pancreatic cell lines . Toxicity to a normal foreskin Hs68 fibroblast cell line was studied as a control. Finally, the extract showing the highest cytotoxicity in all three cancer cell lines was evaluated for its apoptotic activity via a caspase-3 quantification assay.Fifty-six plant extracts Table  commonlyDetails of the extraction process have been described previously . BrieflyStock solutions of the extractives were prepared by dissolution in ethanol. The final concentrations of the extract dilutions in culture were 0.1, 1, 10, and 100 μg/mL. The concentration of ethanol in these dilutions was restricted to no more than 0.5% (v/v) to minimize potential effects of the solvent on cell growth. Doxorubicin hydrochloride (DOX), Curcumin (Cur.), and Staurosporine, used as positive controls, were purchased from Sigma Aldrich .Three human pancreatic carcinoma cell lines and were used in this study: Panc-1, derived from a mostly differentiated carcinoma ; MIA PaC2 in sterile DMEM medium with 10% fetal bovine serum (Panc-1 and Hs68), DMEM with 10% fetal bovine serum and 2.5% horse serum (MIA), and IMDM with 20% fetal bovine serum (Capan-1), after the addition of 4 mM L-glutamine and penicillin-streptomycin. Cell culture media was obtained from the School of Chemical Sciences Cell Media Facility at the University of Illinois at Urbana-Champaign. Cells were grown in standard tissue culture flasks and upon reaching 80% confluence were passaged with a solution of 0.25% trypsin-EDTA.grown at 37°C and 5% COThe preliminary determination of the effect of each plant extract on Panc-1 cells was made in an initial screen using a label-free PC biosensor-based assay. Details of the PC biosensor and imaging instrumentation have been described ,13. BrieTo perform the PC biosensor screening assay, an initial baseline PWV scan was first taken in which each biosensor microplate well contained 25 μL cell culture media. Following this baseline scan, the media was aspirated and the cells were then plated into the wells of a 384-well biosensor microplate at a density of approximately 500 cells/well and allowed to attach overnight at 37°C. To verify cell plating uniformity, a post-attachment scan was taken after cell attachment and the PWV shift image was determined by subtracting the baseline image from the post-attachment image. The cells were then incubated for 24 hours with 25 μL of extract in media at a final concentration of 100 μg/mL. Control cultures and blank wells without cells received 100 μL of medium with 0.5% (v/v) of ethanol. After the 24 h drug exposure period, the cells were grown for an additional 24 h in extract-free fresh medium. Finally, a post-treatment scan was taken and the PWV shift image was determined by subtracting the baseline image from the post-treatment image. Increases in the local PWV as registered by the local pixels were observed only in regions where cells formed a close physical attachment to the sensor surface. Dead cells and other debris that are physically deposited on the same surface did not register similar increases in the local PWV. Estimates of the number of the cells that were attached to the sensor surface in each well were made using a PWV shift threshold as described earlier . Cell coThe MTT assay was used to confirm the results of the initial PC-based screen and test identified leads on three additional cell lines. Changes in proliferation rates of cells treated with various plant extracts were determined using the ATCC MTT Cell Proliferation Assay kit. The assay, previously described , is base4 cells/well and allowed to attach overnight at 37°C. The cells were then incubated with 100 μL of an extract at a concentration of 100 μg/mL for 24 hours. Untreated cultures and blank wells without cells received 100 μL of medium with 0.5% (v/v) of ethanol. After the drug exposure period, the cells were grown for an additional 24 hours in extract-free fresh medium. Next, 10 μL of the MTT reagent was added to each well, and the plate was incubated for 4 hours at 37°C. The MTT crystals were then solubilized overnight by adding 100 μL of the MTT detergent reagent to each well. Absorbance measurements were made at 570 nm using a Biotek® Synergy HT Spectrophotometer. Proliferation was expressed as the fraction of treated cells that survived relative to untreated cultures. The IC50 values were calculated using GraphPad Prism 5.0 . Every experiment included a set of negative controls (untreated cultures) as well as two sets of positive controls (DOX and Curcumin). All experiments were performed in triplicate and repeated at least twice.Cells were plated in 96-well flat bottom tissue culture plates at a density of approximately 1-1.2 × 10p-NA substrate cleaved by cell lysates to release the colored p-NA molecule. Here, PANC-1 cells were exposed to the P. punctata extract (100 μg/mL) or Staurosporine (0.1 μg/mL), a known apoptotic agent. Following treatment, the cells were washed in PBS and suspended in a lysis buffer  for 15 minutes at a concentration of 107 cells per 100 μL of buffer. Lysed cells were centrifuged at 16,000 × g, 4°C for 15 minutes. Lysate protein concentrations were determined using the Bradford assay. Equal amounts of protein (~ 20 μg) from each sample were added to wells containing the assay buffer , followed by 10 μl of Ac-DEVD-p-NA (20 mM), bringing the total volume of each well to 100 μl. Caspase-3 activity was assessed by measuring the optical density at 405 nm using a Biotek® Synergy HT Spectrophotometer. The amount of Ac-DEVD-pNA cleaved was then calculated and plotted. The effect of Ac-DEVD-CHO, a caspase-3 inhibitor, on caspase-3 activity was studied simultaneously. As per the manufacturer's instructions, 10 μl of the inhibitor Ac-DEVD-CHO (2 mM) was added to a well containing the assay buffer, followed immediately by the cell lysate and 10 μl of the substrate Ac-DEVD-pNA (20 mM). All experiments were performed in duplicate and repeated at least twice.Caspase-3 activity was assessed using the caspase-3 Colorimetric Assay Kit (Sigma Aldrich), following the manufacturer's instructions. This assay is based on the detection of the amount of Ac-DEVD-t-test. All statistical analyses were performed using GraphPad Prism 5.0 . Differences were accepted as statistically significant at p < 0.05.Results were expressed as means ± SD of replicates. Comparison between data sets was performed using one way analysis of variance (ANOVA) followed by Student's P. punctata (No. 34) where nearly 100% cell death was observed. Finally, Figure A. glabra (No. 11) where the extract enhanced cell proliferation. Using the thresholding method, the number of cells in each well after the initial seeding period was determined from the top PWV shift image. After the treatment period, cell counts for each well were obtained from the bottom PWV shift image and counts from treated wells were compared against those from untreated control wells present on the same plate. The data from PWV shift images of the 56 extracts were converted into cell survival fractions relative to untreated wells as shown in Figure An initial screen of fifty-six extracts was performed on the Panc-1 cell line using the PC biosensor-based cell attachment assay. Biosensor images of representative individual wells of a 384-well biosensor microplate before and after treatment are shown in Figure P. punctata (No. 34), A. sessilis (No. 4), and A. chittagonga (No. 6) - were found to induce over 70% cell death in all three cell lines. Dose response studies of these three extracts on three cancer cell lines and one fibroblast cell line (Hs68) are presented in Figure 50 values are summarized in Table P. punctata extract had IC50 values of 20.34 μg/mL, 24.97 μg/mL, and 31.39 μg/mL for Panc-1, MIA, and Capan-1, respectively. However, this extract was also toxic to Hs68 cells with an IC50 value of 25.81 μg/mL. Similarly, the A. sessilis (No. 4) extract showed high cytotoxicity to the cancer cell lines with IC50 values in the 13.08 μg/mL - 34.92 μg/mL range but limited selectivity. In contrast, A. chittagonga (No.6) showed high cytotoxicity to all cancer cells (IC50 of 42.8 μg/mL - 49.8 μg/mL) and low cytotoxicity to the fibroblast cells (IC50 > 100 μg/mL)The nine extracts identified in the PC-based screen were then tested using the MTT assay on the Capan-1, and MIA cell lines, in addition to the Panc-1 cells. Four of these extracts  induced over 50% death in the MIA and Capan-1 cell lines  was not selectively cytotoxic to pancreatic cancer cells, we were interested to see if the observed high cytotoxicity to Panc-1 cells treated with P. punctata was mediated via apoptosis. To further elucidate the mechanism of cell death induced by P. punctata, a caspase-3 colorimetric assay was conducted to establish the levels of caspase-3 activation both before and after treatment with the extract. The results of this experiment show that treatment of Panc-1 cells with P. punctata strongly induces increased caspase-3 activity  and Curcumin exhibited the same level of cytotoxicity on Panc-1 cells as measured through an MTT assay. Figure P. punctata was similar to that observed with Staurosporine-treated cells. In contrast, untreated controls were shown to maintain a basal caspase-3 activity  and their expression of multiple drug resistant proteins [Due to the high mortality rates of pancreatic cancer and the absence of effective chemotherapy, there is a continued need for new alternatives for treatment and prevention, and natural products play a dominant role in the discovery of such new drugs ,19. In tproteins -23. Our P. punctata (No. 34), A. sessilis (No. 4), and A. chittagonga (No. 6). While all three extracts showed low cytotoxicity to fibroblast cells at low concentrations (< 20 μg/mL), only A. chittagonga continued to exhibit low cytotoxicity at elevated concentrations  has been associated with potential pharmacological activity. The results of a phytochemical screening of the methanolic extract of P. punctata showed the presence of steroids as an active component [P. punctata has also been shown to possess strong antimicrobial activity [a Figure . Interesomponent . Moreoveomponent . P. puncactivity . In addiactivity ,27.P. punctata, A. sessilis has shown promise in relatively disparate areas of medicine. A member of the Amaranthaceae family, A. sessilis has a history of use in Indian and Chinese traditional medicine [A. sessilis in cancer treatment has been reported.Similar to medicine . Preparamedicine . A recenmedicine ,29. HoweAmoora chittagonga belongs to the Meliaceae family. A few Amoora species have been shown to possess anti-inflammatory, anti-cancer, and hepatoprotective medicinal properties [A. rohituka) has been shown to induce apoptosis in breast carcinoma through caspase activation [A. chittagonga. We found that A. chittagonga showed anti-proliferative properties in several models of pancreatic cancer, while maintaining low toxicity to non-cancerous Hs68 fibroblast cells.Finally, operties . The comtivation . Furthertivation . HoweverWhile not the primary focus of this work, it is worthwhile to discuss some of the advantages afforded by the PC biosensor system in our cell-based screening assays. Since these biosensors are incorporated into microplates, they can be used with standard microplate liquid handling instrumentation such as pipetting systems and wash stations. Furthermore, assays using as few as 500 Panc-1 cells per well in a 384-well plate can be performed, which is particularly important for cell lines that proliferate slowly. A comparable colorimetric based MTT proliferation assay requires 2500-3000 cells per well. Although the assays shown in this work utilize cells that readily attach to the biosensor surface without the use of specific capture molecules, previous work has also demonstrated the use of PC biosensors for cells that would ordinarily remain suspended in solution by preparing the biosensor with immobilized proteins that specifically bind with outer membrane proteins . We have® from Promega, WI, USA) uses a single reagent to perform both the cell lysis and the caspase cleavage of a substrate leading to the generation of luminescence, which is detectable using any standard microplate-compatible spectrophotometer. Such additional interrogation allows much more information to be derived from a single culture of cells, thereby decreasing the role of biological variability on the results of a given trial, experiment, or screening assay.Since PC biosensor assays are label-free, cells are not stained, killed, or altered in any way. Hence, it is possible to use the biosensor to perform proliferation assays, and to subsequently use the same cells to interrogate the presence of a protein, such as caspase-3. Caspase-3 protein detection and quantification can be performed using commercially available kits designed to work directly with cultures of adherent cells. One such test (Caspase-GloP. punctata, A. sesselis, and A. chittagonga often provide excellent starting points, deriving the mechanistic advantages of such compounds will allow the development and realization of specific, highly potent anti-cancer drugs with limited peripheral toxicity.In this work, we screened 56 extracts of 44 unique Bangladeshi medicinal plants for their anti-proliferative properties in three cultured pancreatic cancer cell lines. Screening of each of the extracts identified three extracts with the desired properties. Further testing revealed one extract to induce high toxicity to multiple cancerous cell lines while preserving relatively low levels of toxicity toward a non-cancerous control cell line. Our results provide the basis for the further investigation of each of these species and potential identification of novel bioactive compounds with therapeutic, anti-cancer properties. Elucidating the mechanisms by which these anti-cancer properties are derived from the plant extracts is of crucial importance. Such clarification is necessary to identify, select for, and optimize therapeutic compounds. Though naturally occurring compounds such as those found in Brian T. Cunningham discloses that he is a founder of SRU Biosystems, who provides PC biosensors and detection instruments on a commercial basis.SG performed the PC assays, MTT assays, and analyzed the resulting data. SG, SVB, and EAL performed the caspase-3 assays and analyzed the data. SG, BTC, EAL, and KLW wrote and reviewed the manuscript. ISA, AA, BTC, and KLW are co-investigators of this project. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6882/10/52/prepub